Buffer system for increasing seroconversion efficiency

ABSTRACT

A method of increasing efficiency of deantigenation of blood group epitopes on erythrocytes (seroconversion) by exoglycosidases utilizing a step of performing the deantigenation in a zwitterionic buffer. The method provides a buffer system and exoglycosidase that utilizes a twenty to one hundred fold lower total enzyme mass than the prior art methods.

TECHNICAL FIELD

The present invention relates to a purified enzyme preparation of thetype used in the conversion of certain sub-types of blood typeerythrocytes to type O cells to render the cells useful for transfusiontherapy. More specifically, the present invention provides a novelbuffer system rendering the process commercially useful.

BACKGROUND OF THE INVENTION

The A, B, and H antigens are a clinically significant blood group(Landsteiner, 1901; Mollison et al, 1987). These antigens are terminalimmunodominant monosaccharides on erythrocyte membrane glycoconjugates(Harmening, 1989). High densities of these epitopes are present onerythrocyte membranes and antibodies bound to these antigens readily fixcomplement (Economidou, et al, 1967; Romano and Mollison, 1987). Becausethese epitopes are ubiquitous in nature, immuno-potent and naturallyoccurring, complement fixing antibodies occur in individuals lackingthese antigens, and transfusion of incompatible blood results in fatalhemolytic transfusion reactions (Fong et al, 1974; Schmidt, 1980).

Complex sugar chains in glycolipids and glycoproteins have often beenimplicated in the growth and development of eukaryotes (Watanabe et al.,1976). In particular, complex sugar chains play an important part in therecognition of self in the immune system (Mollison et al., 1987).Exoglycosidases are enzymes which can modify carbohydrate membraneepitopes, thereby modulating the immune response (Goldstein et al.,1982). The α-D-galactosidase from Glycine max is an enzyme that degradesthe human blood group B epitope to the less immunogenic blood group Hantigen also known as blood group O (Harpaz et al., 1977).

α-D-galactosidases [EC 3.2.1.22] are a common class of exoglycosidases.Although physical properties of these enzymes differ as a group, and thephysiological significance of these enzymes are not clearly established,isozymes of α-D-galactosidase are common to many plant species (Flowerset al, 1979; Corchete, et al 1987). Several investigators have studiedα-D-galactosidase from Coffea (Yatziv, 1971). There are reports thatseveral isozymes exist for the Coffea α-D-galactosidase enzyme(Courtois, 1966).

Modification of the A, B, and H antigens using exoglycosidases tohydrolyze the terminal immunodominant residue has previously beendescribed (Tsuji et al, 1990; Levy & Aminoff, 1978; Yatziv & Flowers,1971; Kubo, 1989). Hydrolysis of the terminal N-acetyl-α-D-galactosamineby α-N-acetyl-galactosaminidase (EC 3.2.1.49) converts blood type A₂ toblood type O, and similarly, hydrolysis of the terminal α-D-galactoseresidue by α-D-galactosidase (EC 3.2.1.22) converts blood type B to O(Yatziv & Flowers, 1971; Levy & Aminoff, 1978). An α-D-galactosidasefrom Coffea canephora has been shown to effectively convert type Berythrocytes to type O erythrocytes (Harpaz, 1975). Because type Oerythrocytes are generally universally transfusable, enzymaticdeantigenation would have important medical applications.

Improvements of this technology could increase the compatible bloodsupply while reducing waste and risk of transfusion reactions. Theprimary impediments to seroconversion have been the large quantities ofenzyme required for deantigenation, and washing the red cellconcentrates to achieve the desired pH (Goldstein, 1989). Further, thereaction needs to take place at 24° C. Standard transfusion medicineprotocol requires treating erythrocytes at or below 24° C. in order todecrease the possibility of bacterial contamination and maintain cellfunction and survival. Therefore, it is commercially important toisolate enzymes and develop buffer systems in which efficientseroconversion can occur at 24° C.

Work by Goldstein et al., 1982, lead to the feasibility of large scaleenzymatic conversion of blood type B to O erythrocytes (Lenny et al,1982, 1991). This group used Coffea α-D-galactosidase in PCBS buffer toachieve deantigenation. These cells were transfused into individualswith anti-B antibodies and survived normally. The current problem withthis application is that very high enzyme concentrations, about one totwo grams of exoglycosidase per transfusable unit of red cells, arerequired for deantigenation (Lenny and Goldstein, 1991). The cost ofthis amount of enzyme is enormous and, without reduction, renders thistechnology impractical.

Data establishing the optimal ionic strength, pH, buffer species, orenzyme concentration for efficient deantigenation has not beenpublished. It is presently unknown whether exoglycosidase activity canbe modified to achieve more efficient hydrolysis of the B antigen in redcell concentrates.

U.S. Pat. No. 4,330,619, issued May 18, 1982; U.S. Pat. No. 4,427,777,issued Jan. 24, 1984; and U.S. Pat. No. 4,609,627, issued Sep. 2, 1986,all to Goldstein, relate to the enzymatic conversion of certainerythrocytes to type O erythrocytes. The above-mentioned U.S. Pat. Nos.4,330,619 and 4,427,777 disclose the conversion of B-type antigen toH-type antigens by using α-D-galactosidases from green coffee beans(Coffea canephora). The patent discloses the significant potential ofsuch enzymes to be used in the conversion of type B erythrocytes to typeO erythrocytes but does not provide a commercially feasible method.Additionally, other compounds such as tannins, present inα-D-galactosidase enzyme isolates from plants such as Coffea beans canpotentially inhibit or impair enzyme function which provides a furtherdisadvantage for their commercial use (Goldstein et al, 1965).

It would also be useful to have additional exoglycosidases, particularlythose active at neutral pH, that could be used in the deantigenation ofblood group serotypes for transfusions. However, the screeningprocedures currently available to undertake a survey of procaryoticspecies that produce exoglycosidases active at neutral pHs against bloodgroup epitopes (Tsuji et al., 1990; Aminoff & Furukawa, 1970; Levy &Aminoff, 1980) and to characterize the resulting cells are cumbersome,time consuming and expensive to run.

For example, quantitation of red cell membrane deantigenation has beenaccomplished by conventional hemagglutination assays. However,hemagglutination titers are not highly sensitive and are technicallycumbersome. Furthermore, a 50% decrease in antibody concentration onlycorrelates with a one-fold change in titer. Thus, it is difficult tovary a large number of parameters and detect subtle changes indeantigenation using this assay.

A sensitive, rapid assay that could be used in deantigenation studies onnative red blood cells and could be used for screening culture banks orselecting bacterial mutants that constitutively express blood groupspecific enzymes would be very useful. It would also be useful if theassay could be used to characterize other blood group specificexoglycosidases as well as blood group A activeα-N-acetyl-galactosaminidases and blood group systems I and P whichexpress terminal immunodominant saccharide epitopes.

Finally, for further studies of the soybean exoglycosidase and for moreefficient production and supply of purified soybean α-D-galactosidase,it would be useful to have recombinant soybean α-D-galactosidaseavailable. Cloning of the gene will allow rapid and efficient productionof the enzyme.

SUMMARY OF THE INVENTION AND ADVANTAGES

According to the present invention, a method of increasing efficiency ofdeantigenation of blood group epitopes on erythrocytes (seroconversion)by exoglycosidases utilizing a step of performing the deantigenation ina zwitterionic buffer. The method provides a buffer system andexoglycosidase that utilizes about twenty to one hundred fold lowertotal enzyme mass than the prior art methods with an enzyme isolatedfrom Coffea.

When deantigenating the ABO blood type, the method includes the steps ofisolating A, B, or AB erythrocytes and suspending the isolatederythrotypes in an enhancing zwitterionic buffer. The exoglycosidase isthen added and the cell suspension is incubated at 24° C. for betweenone and two hours and washed in phosphate buffered saline. This methodprovides an economically viable means for seroconversion of A and Berythrocytes to be used in transfusion thereby increasing the compatibleblood supply while reducing waste and risk of transfusion reactions.

A flow cytometry assay has been developed for identifying and studyingblood group specific exoglycosidases. The flow cytometry assay can beused to identify optimal deantigenation conditions with objectivity.With this assay cells from larger scale assays can be harvested andtheir morphology/structure and function characterized. The advantages ofthis assay include the use of native cells, the ability to perform largenumbers simultaneously, and the ability to easily determine enzymeactivity over a variety of conditions.

Finally, the present invention provides the DNA (SEQ ID No.:2) and aminoacid (SEQ ID No.:4) sequences of Glycine max α-D-galactosidase.

BRIEF DESCRIPTION OF THE DRAWINGS

Other advantages of the present invention will be readily appreciated asthe same becomes better understood by reference to the followingdetailed description when considered in connection with the accompanyingdrawings wherein:

FIG. 1 is a bar graph of the agglutination score following Glycine maxα-D-galactosidase deantigenation of type B erythrocytes in threebuffers, PCBS, GCB and CBS, all data points are the means of sixindependent determinations;

FIG. 2 is a graph of the agglutination score following Glycine maxα-D-galactosidase deantigenation of type B erythrocytes as a function ofNaH₂ PO₄ (open square) or Na citrate (filled diamond) concentrations (0,1.25, 2.50, 5, 10, 20, 40), all data points are the means of sixindependent determinations; data points initially overlap;

FIG. 3 is a graph of the agglutination score following Glycine maxα-D-galactosidase deantigenation of type B erythrocytes as a function ofNaCl concentration (0, 5, 10, 20, 40), all data points are the means ofsix independent determinations;

FIG. 4 is a graph of the agglutination score following Glycine maxdeantigenation of type B erythrocytes as a function of glycine and NaClconcentration (300,0), (240,30), (180,60), (120,90), (60,120), (0,150),all data points are the mean of six independent determinations;

FIG. 5 is graph of the agglutination score following Glycine maxα-D-galactosidase deantigenation of type B erythrocytes as a function ofpH (5.4, 5.6, 5.8, 6.0, 6.2, 6.6, 6.8, 7.0, 7.2), all data points arethe mean of six independent determinations;

FIG. 6 is a graph of the agglutination score following Glycine maxα-D-galactosidase deantigenation of type B erythrocytes as a function ofhematocrit percentage (8,16,24,32), all data points are the mean of sixindependent determinations;

FIG. 7 is a graph of the agglutination score following Glycine maxα-D-galactosidase deantigenation of type B erythrocytes as a function ofenzyme concentration (1.25, 2.5, 5.0, 10.0, 20.0) and hematocrit of 8%(open square) or 16%, (filled diamond), all data points are the mean ofsix independent determinations;

FIG. 8 is a graph of the agglutination score following Glycine maxα-D-galactosidase deantigenation of type B erythrocytes as a function oftime (0.25, 0.5, 1.0, 2.0), all data points are the mean of sixindependent determinations;

FIGS. 9A-B are photomicrographs of the morphology and agglutination ofGlycine max α-D-galactosidase deantigenated type B erythrocytes anduntreated erythrocytes, Panel A: enzyme treated erythrocytes; Panel B:untreated erythrocytes;

FIG. 10 is a bar graph of the reactivity of type A, B, and Oerythrocytes with monoclonal anti-B, all data points are the means ofthree independent determinations;

FIG. 11 is a graph of percent of FITC labelled cells as a measurement ofdeantigenation as a function of enzyme concentration (0.32, 0.63, 1.25,2.50, 5.0, & 10.00), all data points are the means of three independentdeterminations;

FIG. 12 is a graph of percent of FITC labelled cells as a measurement ofdeantigenation as a function of time (0, 15, 30, 60, 120 minutes), alldata points are the means of three independent determinations;

FIG. 13 is a graph of percent of FITC labelled cells as a measurement ofdeantigenation as a function of pH (5.4, 5.8, 6.2, 6.6, 7.0, 7.4), alldata points are the means of three independent determinations;

FIG. 14 is a bar graph of percent of FITC labelled cells as ameasurement of deantigenation as a function of buffer composition (PCBS,GCB, CBS), all data points are the means of three independentdeterminations; and

FIG. 15 is a graph of percent of FITC labelled cells as a measurement ofdeantigenation as a function of glycine and NaCl concentration (0,129),(52,103), (103,77), (173,52), (206,26), (258,0), all data points are themeans of three independent determinations.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

The present invention provides a method of increasing efficiency ofdeantigenation of blood group epitopes on erythrocytes, seroconversion,by exoglycosidases. Generally, the method includes a step of performingdeantigenation in an enhancing buffer.

In a preferred embodiment, when deantigenating either B or A epitopesusing α-D-galactosidase from Glycine max and Gallus domesticusα-N-acetyl-galactosaminidase, respectively, the method generallyincludes the steps of isolating A, B, and AB erythrocytes and suspendingthe isolated erythrocytes in a zwitterionic buffer. The appropriateexoglycosidase is then added and the cell suspension incubated at 24° C.for between one and two hours and washed in phosphate buffered saline toremove both the exoglycosidase and enhancing buffer.

Additionally other blood types with a galactose or N-acetylgalactosamineterminal group such as P₁ can be modified with the present invention.

The exoglycosidases are selected from the group consisting of Glycinemax α-D-galactosidase, Gallus domesticus α-N-acetyl-galactosaminidase,Phaseolus vulgaris α-galactosidase and other multimeric eucaryoticexoglycosidases containing multiple subunits. Monomeric enzymes, i.e. anenzyme without subunits, may also be enhanced.

The zwitterionic buffer contains zwitterions selected from the groupconsisting of glycine, alanine, CHAPS, and zwitterions not containingadditional charged groups, i.e., neutral overall charge. The glycine isused at a 220 to 440 mM concentration, in a buffer having 0.1 to 20 mMNa citrate, and 0.01 to 30 mg ml⁻¹ albumin at pH 5.8. In a preferredembodiment, the zwitterionic buffer consists of 5 mM Na citrate, 300mMglycine and 1 mg ml⁻¹ albumin at pH 5.8. In a further preferredembodiment; the albumin is human serum albumin.

In determining the enhancing buffer, the type of exoglycosidase isconsidered. The subclass of exoglycosidases including α-D-galactosidasefrom Glycine max (SEQ ID No.:4) and Gallus domesticusα-N-acetyl-galactosaminidase respond to zwitterionic buffers.

The pH of the buffer is generally effective between 5.4 and 6.4 with apH of 5.8 as the preferred embodiment. Deantigenation was done at ahematocrit between 8% and 16%.

In a preferred embodiment, α-D-galactosidase from Glycine max and Gallusdomesticus α-N-acetyl-galactosaminidase were used for deantigenation inan enhancing buffer which contained zwitterions. The optimalconcentrations of the zwitterions were determined as described inExamples 2 and 3 hereinbelow. For glycine, a concentration of 220 to 440mM was found to be optimum.

The suitability of α-D-galactosidase from Glycine max over Coffeaα-D-galactosidase was shown by the following data. Lower concentrationsof α-D-galactosidase from Glycine max, as low as 2.0 U ml⁻¹, completelyremoved B antigen from native erythrocytes in GCB buffer at lowhematocrits in cell suspension assays. However, under similarconditions, the Coffea canephora enzyme activity was undetectable. Bothenzymes had similar activities in the PCBS buffer, but, because theGlycine max enzyme has a much higher specific activity, a smaller massof enzyme was required to achieve deantigenation in PCBS buffer.

Inhibition of enzymatic activity at physiologic ionic strength (μapproximately 0.130 to 0.145) was significant, however, isosmoticconcentrations of the zwitterion glycine did not inhibit the Glycine maxenzyme, whereas the Coffea enzyme activity was not enhanced.

With the present invention, deantigenation with Glycine max enzyme, atlow hematocrits, can be achieved at higher pHs in the GCB buffer, closerto physiologic conditions. The active pH range of the Glycine enzyme wassuitable for enzymatic conversion. The pH used in the deantigenationbuffer system has been shown to provide recovery of viable, transfusablecells (Goldstein et al., 1987; Goldstein, 1989).

Glycine containing buffers enhanced enzyme activity on nativeerythrocyte membranes. Other zwitterions had a similar effect. It isknown that glycine disrupts the ion cloud around red cell membranesreducing the zeta potential (Issit, 1985). From the soluble Btrisaccharide substrate studies, it was apparent that activity in GCBwas similar to PCBS. This implies that glycine must somehow alter theinteraction of the Glycine max α-D-galactosidase with the erythrocytemembrane enhancing hydrolysis of the B epitope. This is supported bydata which shows that the effect of glycine is diminished withincreasing NaCl concentrations.

The safety of use of glycine in a cell preparations to be used in humanswas considered. Although glycine is a known neurotransmitter, the meanresidual glycine concentration measured in the final wash buffers, 2.43μM, was below the reported physiologic concentration of glycine, median275 μM, range 120 to 386 μM (Issit, 1985) ands therefore, would not posea problem. Further, the present invention will not provide levels ofglycine where physiologic reactions have been described (Sherwood et al,1991; Mizutani et al, 1990) .

The step of incubating requires that the enzyme be active at 24° C. Theenzyme isolated from Glycine max was active at 24° C., only slightlyless than at 37° C. Standard transfusion medicine protocol requirestreating erythrocytes at or below 24° C. in order to decrease thepotential of bacterial contamination and maintain cell function andsurvival.

In GCB buffer, red cell function was unchanged when measured by methodsdescribed hereinbelow. The only detected antigenic change was the B andP epitopes in cell phenotyping. similar results have been reported forPCBS buffer with Coffea canephora α-D-galactosidase (Goldstein et al,1982).

Interestingly, cells incubated in PCBS buffer developed spicules asdescribed by others (Goldstein, 1982). However, cells incubated in GCB,however, maintain normal cell morphology.

One of the major obstacles to seroconversion technology is the enormousquantities of enzyme required for deantigenation; approximately one totwo grams of purified Coffea enzyme is required to deantigenate the Bepitope from one unit of packed red blood cells (Goldstein et al, 1982;Goldstein, 1989). By using Glycine max α-D-galactosidase in azwitterionic buffer, deantigenation at about a twenty to one hundredfold lower total enzyme mass can be achieved than with a purified Coffeaenzyme. This buffer-enzyme combination is an economically feasiblealternative to the Coffea enzyme in PCBS.

Further, purification from plants of exoglycosidases can co-isolatecontaminants that are harmful and that are expensive to remove such asin Coffea extractions which contain tannins (Goldstein et al, 1965). Thepresent invention provides the sequence of the Glycine maxα-D-galactosidase (SEQ ID Nos:2,4) so that a purified preparation ofrecombinant enzyme is available.

In an additional experiment, activity of a Gallus domesticus enzyme onerythrocyte membranes was modified by different buffer species. Maximalhydrolysis of the A epitope on red cell membranes was seen when using aPGB buffer. This further shows that glycine alters the enzyme-membraneinteraction and that zwitterionic buffers can improve enzyme efficiencyin deantigenation.

Coffea α-D-galactosidase has been used in the prior art forseroconversion of B epitopes on erythrocytes, but a zwitterionic bufferwas not effective in increasing its efficiency.

Applicants have developed a novel procedure (co-pending application U.S.Ser. No. 07/996,029 incorporated herein by reference) for thepurification of the Coffea α-D-galactosidase enzyme which results in aproduct with a specific activity of 145.7 U mg⁻¹ min⁻¹ which is higherthan the 25 U mg⁻¹ min⁻¹ value previously described by others (Haibachet al., 1991; Lenny et al, 1982). Hydrolysis can be enhanced compared toPCBS by performing hydrolysis in 10mM MES or Na citrate+140 mM NaCl atPh 5.8. This enhancing buffer provides a two-fold increase inefficiency, MES enhancement of B epitope hydrolysis was mirrored in thesoluble phase carbohydrate studies suggesting that this compound affectsthe enzyme rather than the erythrocyte membrane.

These findings show that increases in exoglycosidase efficiency can beachieved with changes in buffer systems.

In undertaking the above experiments, it was useful to vary a largenumber of parameters and detect subtle changes in deantigenation. Asensitive, rapid flow cytometry assay that can be used in deantigenationstudies on native red blood cells and can be used for screening culturebanks or selecting bacterial mutants that constitutively express bloodgroup specific enzymes was developed. The method includes the steps ofpreparing erythrocytes in suspension and adding an exoglycosidase undera variety of buffer conditions and concentrations. Following incubation,the cells are labeled and the deantigenation efficiency monitored with aflow cytometer.

It is a useful assay that can also be used to characterize other bloodgroup specific exoglycosidases as well as blood group A activeα-N-acetyl-galactosaminidases and blood group systems I and P whichexpress terminal immunodominant saccharide epitopes and only require thesubstitution of the appropriate antibodies specific for the blood groupbeing assayed.

The flow cytometry assay of the present invention can be used toidentify optimal deantigenation conditions with sensitivity andobjectivity. Furthermore, cells from larger scale assays can beharvested and their morphology/structure and function characterized.

The Coffea canephora α-D-galactosidase currently used for deantigenationof native erythrocytes has an acidic pH optima (Kadowaki et al, 1989;Courtois & Petek, 1966). Numerous procaryotic species produceexoglycosidases active at neutral pHs against blood group epitopes(Tsuji, et al, 1990; Aminoff & Furukawa, 1970; Levy & Aminoff, 1980).Additionally, many procaryotic exoglycosidases are active againstglycolipid and glycoprotein blood group epitopes and inactive againstlow molecular weight chromogenic substrates (Hoskins et al, 1987). Moretraditional assays such as mucin or glycolipid hydrolysis followed byquantitation of liberated monosaccharide are cumbersome and timeconsuming. The flow cytometry assay is ideal for sensitivedeantigenation studies on native red blood cells and can be used forscreening culture banks or selecting bacterial mutants thatconstitutively express blood group specific enzymes. This assay can alsobe used for the characterization of other blood group specificexoglycosidases as well as blood group A activeα-N-acetyl-galactosaminidases and blood group systems I and P whichexpress terminal immunodominant saccharide epitopes.

Flow cytometry assays can be employed to characterize enzymaticmodification of these epitopes. The advantages of this assay include theuse of native cells, the ability to perform large numberssimultaneously, and the ability to easily determine enzyme activity overa variety of conditions.

The above discussion provides a factual basis for the use of thezwitterionic buffer. The methods used with and the utility of thepresent invention can be shown by the following examples.

EXAMPLES

Reagents

The source of reagents is as follows: Immulon 4 flat bottom microtiterwells (Dynatech Laboratories, Chantilly, Va.), murine monoclonal anti-Band monoclonal anti-A (Ortho Diagnostics, Raritan, N.J.), goatanti-murine μ-chain specific alkaline phosphatase conjugate (Calbiochem,LaJolla, Calif.), carbohydrate substrates (Accurate Chemicals, Westbury,N.Y.), lectins (EY Laboratories, San Mateo, Calif.). Biotinylated Ulexeuropaeus type I (UEA I) and Dolichos biflorus (DBA) lectins werepurchased from EY Laboratories, San Mateo, Calif.

Other reagents were obtained as follows: bovine serum albumin (BSA),human serum albumin (HSA) deoxycholic acid, cetylpyridinium chloride,CHAPS, Triton X-100, 2-[N-Morpholino]ethanesulfonic acid (MES),p-nitrophenyl phosphate tablets, and alkaline phosphatase conjugatedavidin (Sigma Chemical Co., St. Louis, Mo.), Bradford reagent (Bio-Rad,Hercules, Calif.), BCA reagent (Pierce Chemical Company, Rockfield,Ill.). Columns for gas chromatography were purchased from Quadrex, NewHaven, Conn. Solvents were purchase from Aldrich Chemical Company,Milwaukee, Wis., and distilled prior to use. Carbohydrates employed aschromatography standards were purchased from Pfanstiehl Laboratories,Inc., Waukegan, Ill. All other chemicals were purchased from FisherScientific, Pittsburgh, Pa.

Polyclonal antisera reacting to Glycine max α-D-galactosidase wasprepared in rabbits by standard methodologies (Harlow & Lane, 1988). Therabbit antisera prepared to Glycine max lectin did not react withGlycine enzyme in an ELISA.

Volumes (0.9) of native human type erythrocytes were collected in 0.1volumes of 3.2% Na citrate and stored at 4° C. prior to use. Diagnostickits for ATP, 2,3 DPG, and cholinesterase were purchased from SigmaChemical Co., St. Louis, Mo.

Enzyme Preparations

Gallus domesticus α-N-acetyl-galactosaminidase was purified aspreviously described (Hata et al, 1992).

Coffea canephora isozyme was purified as previously described (Haibachet al., 1991). Its mean specific activity was 145.7 U mg⁻¹ min⁻¹ and washomogeneous by SDS PAGE.

Bos α-L-fucosidase was purified by a modification of the method ofSrivastava et al, 1986. Final purification of the enzyme was purifiedusing the affinity ligand α-L-fucopyranosycamine.

Glycine max α-D-galactosidase was purified by a modified procedure ofHarpaz et al., 1975. Enzyme activity was measured as previouslydescribed with one unit (U) defined as one μmole of substrate hydrolyzedper minute (Haibach et al, 1991. The preparations had mean specificactivities in the range of 194-213 U mg⁻¹ min⁻¹ and were homogeneous bySDS PAGE according to the method of Laemmli (1970). No hemagglutinins totype A, B, or O erythrocytes were detected in the preparations.

General Methods

Protein concentration was determined by the method of Bradford (1976).

ELISA Methodology for A₂ Membranes

The erythrocyte membrane preparation procedure, plate coating technique,and ELISA method are described by Hobbs et al. (1993), with the onlydifferences being the use of A₂ erythrocyte membranes and monoclonalanti-A. Briefly, microtiter wells were coated with A₂ membranes,exoglycosidase treated, probed with anti-A IgM monoclonal antibody, thendeveloped with anti-murine μ-chain specific alkaline phosphataseconjugate. The conversion of the A₂ antigen to the H antigen wasquantitated using the H antigen specific Ulex europeaus type I lectin(UEA I) as previously described, with the exception that the UEA Iconjugate was diluted 1:1600 (Hobbs et al., 1993). Studies wereperformed on A₁ membranes using essentially the same procedures,however, the plates developed with anti-A₁ were incubated with substratefor 15 minutes.

ELISA Methodology for B Membranes

The erythrocyte membrane preparation, plate coating technique, and ELISAmethod used are that of Hobbs et al. (1993). Briefly, microtiter wellswere coated with B Membranes, α-D-galactosidase treated, probed with IgMmonoclonal antibody, then developed with anti-murine μ-chain specificalkaline phosphatase conjugate followed by p-nitrophenyl phosphatesubstrate.

Cell Suspension Studies

Fresh human erythrocytes were washed five times with the indicatedbuffer. The washed cells were diluted to the desired hematocrit in thedescribed buffers, enzyme added, incubated at 24° C. for the determinedinterval, washed five times with PBS (13 mM NaH₂ PO₄ +137 mM NaCl, pH7.4), and assayed by a conventional hemagglutination assay as previouslydescribed (Bryant, 1982). Suspensions were also observed microscopicallyfor hemagglutination and cell morphology. Microscopic aggregates,regardless of size, were assigned a score of 0.5 agglutination units.

Red Cell Structure and Function Studies

Erythrocyte 2,3 DPG, ATP, and cholinesterase were determined aspreviously described (Rose & Liebowitz, 1970; Adams, 1963; Dietz et al,1973). The MCHC (mean corpuscular hemoglobin concentration), MCV (meancorpuscular volume), MCH (mean corpuscular hemoglobin), and RDW (redcell distribution width) were determined on a Coulter STKR by standardlaboratory methods. Osmotic fragilities were determined by the method ofDacie et al. (1938). Methemoglobin and O₂ saturation were determined ona Model 270 Ciba Corning Cooximeter. pO₂ and P50 values were quantitatedwith a Ciba Corning arterial blood gas analyzer. Phenotyping for theABO, CDEce, Kk, Fy^(a) Fy^(b), Jk^(a) Jk^(b), MNSs, Le^(a) Le^(b), andP₁ blood group antigens was performed as previously described(Goldstein, 1989). Glycine was quantitated in the final washed cellsupernatants with a Beckman amino acid analyzer as previously described(Moore & Stein, 1954).

Soluble Carbohydrate Substrate Studies

Reactions contained X μg of substrate and 4.0 μg of enzyme in 120 μl ofCBS (10 mM Na citrate +140 mM NaCl, pH 5.8), PCBS (60mM NaH₂ PO₄ +25 mMNa citrate+75 mM NaCl, pH 5.8), MBS (20 mM MES+140 mM NaCl, pH 5.8), PGB(20 mM NaH₂ PO₄ +140 mM glycine, pH 5.8), or MGB (20 mM MES+140 mMglycine, pH 5.8). Reactions were incubated at 37° C. for the indicatedtime and terminated by increasing the pH to 9.0 and snap freezing.Liberated N-acetyl-D-galactosamine was extracted, derivatized, andquantitated by gas chromatography as described by Mawhinney et al.(1986). Spectra for all extracted carbohydrate derivatives were obtainedand verified against standards on a Kratos MS 50 S mass spectrometerinterfaced with a Carlo Erba Model 4160 gas chromatograph. Mass spectrawere recorded at 70 eV with an ionization current of 50 μA, a sourcetemperature of 250° C., and a transfer temperature of 218° C.

cDNA Preparation and Sequencing

The lambda ZAP::SB cDNA library obtained from Joe Polacco (University ofMissouri) was made using RNA from germinated Williamson's soybeans. Lowstringency hybridization of this library was carried out in a 25%formamide buffer at 42° C., and washes were done in a low stringencywash buffer at 42° C. (Moran & Walker, 1993). High stringencyhybridization of the lambda ZAP::SB cDNA library was done in a 50%formamide buffer at 42° C. (Sambrook et al, 1989), and washes were doneat 65° C. in 0.1% SDS and 0.2×SSC.

Nucleotide probes were made by first purifying the DNA fragment on a 1%low melting point agarose gel (FMC) as described by Maniatis (Sambrooket al., 1989). The DNA fragment was excised from the gel and extractedwith phenol. Excess agarose was precipitated by addition of 1/10 volumeof 4 M LiCl/10 mM EDTA, a ten minute incubation on ice, andcentrifugation at 13,200 RPM in a cold microfuge. DNA was then ethanolprecipitated from the aqueous phase. The purified DNA fragments wereradiolabeled with α³² P by the random primer method (Feinberg &Goldstein, 1983), and the labeled DNA was passed through a spun column(Sambrook et al., 1989) to separate the probe from unincorporatednucleotides. For hybridization, approximately 10⁶ cpm of labeled probewere used per ml of hybridization solution.

Preparative restriction digests were performed using 10 to 20 μg of DNAwith 20 units of enzyme under conditions recommended by the supplier ofthe enzyme.

Plasmid mini and midi preps were performed by alkaline lysis accordingto Maniatis (Sambrook et al., 1989). DNA sequencing reactions were donewith Sequenase 2.0 (USB) under conditions recommended by themanufacturer. Reactions were subjected to electrophoresis on a 6%denaturing acrylamide gel. PCR was performed using the enzyme AmpliTaq,under conditions recommended by the supplier (Cetus). The annealing stepwas carried out at a temperature approximately 5° C. below the lowest Tmfor either primer in the reaction. PCR products were digested, purifiedover a LMP agarose gel, and isolated as described above for thepurification of probe DNA.

Ligations were performed according to standard procedures (Sambrook etal., 1989) using T4 DNA ligase (Promega). Transformations of frozencompetent cells were performed according to the method of Hanahan(1980). Electrotransformations were done according to the Bio Rad GenePulser manual, using approximately 100 ng plasmid DNA or one half of astandard ligation reaction. Prior to electrotransformation, DNA in thecomplete ligation reaction was ethanol precipitated, thoroughly washedtwice with 70% ethanol, and resuspended in 5 μl of water. SK vectorswere transformed in E. coli DH5α and the Pichia vectors were transformedinto E. coil TOP 10 F' which was provided with the Pichia Expression Kit(Invitrogen).

The Pichia Expression Kit was used according to manufacturer'sinstructions and, whenever possible, its suggestions for highestefficiency and expression levels were followed. Constructs in the Pichiaexpression/secretion vectors pHILS1 and pPIC9 were linearized fortransformation with the restriction enzymes Aat II and Tth 111I. Bothvectors have unique Bgl II sites for this purpose, however when thecloned insert contains a Bgl II site it is necessary to use other enzymesites to accomplish linearization. Transformations were done accordingto the recommended spheroplast method in order to maximize probabilityof obtaining transformants containing multiple insertions of the clonedgene of interest. Screening of His+ transformants for the desiredinsertion event, and small and medium-scale growth and induction wereall performed according to the Invitrogen manual provided with the kit.

EXAMPLE 1 DEANTIGENATION OF B EPITOPE WITH COFFEA ENZYME

ELISA studies

Microtiter wells were coated with excellent reproducibility. Wellsdeveloped without enzyme treatment had a mean OD₄₁₀ of 1.480 and aninterplate coefficient of variation of 9.92%. The binding of anti-B as afunction of enzyme concentration decreased as enzyme concentrationincreased. Binding of the H antigen specific lectin from Ulex europaeusincreased with increasing enzyme concentration demonstrating theconversion of B to H antigen.

The effect of different buffer species on Coffea enzyme activity wasstudied. Enzyme activity decreased with increasing buffer concentrationfor all buffer species tested (including Na acetate and Bis-Tris). At50mM only MES produced a noteworthy increase in enzyme activity overPCBS. Similar effects were seen when 140 mM NaCl was buffered with MES.Upon further experimentation, it was determined that MES effectivelyenhanced enzymatic activity over a pH range of 4.5 to 6.0. Increasingconcentrations of Na₂ SO₄ and NaCl inhibited enzymatic activity. Enzymeactivity was inhibited by NaCl concentrations greater than 50 mM. Theeffect of several divalent cations on enzyme activity was studied,including CaCl₂, MgCl₂, MnCl₂, and ZnCl₂. It was found these had neitherinhibitory nor enhancing effects upon enzyme activity.

The effect on enzyme activity of seven different high molecular weightpolymers was determined:poly-L-lysine, poly-L-glutamate, poly-L-alanine,DEAE-dextran, dextran sulfate, dextran, and polyethylene glycol. Eachpolymer had either an inhibitory effect or no effect at the maximalconcentrations tested, 1 mg ml⁻¹.

The activity of the enzyme in the presence of several detergents wasdetermined: deoxycholic acid and 1-pentanesulfonic acid, both anionicdetergents, cetylpyridinium chloride, a cationic detergent, CHAPS, azwitterionic detergent, and n-octyl-β-D-glucopyranoside, a non-ionicdetergent. Enzyme activity in the presence of these detergents wassignificantly decreased.

Coffea α-D-galactosidase activity decreased with increasingconcentrations of glycine. Enzyme activity in the presence of variousconcentrations of galactose dehydrogenase (0 to 5.0 U ml⁻¹) or 2'fucosyllactose (0 to 1.0 mg ml⁻¹) was determined, with both compoundsinhibiting enzyme activity. Enzyme treatment in the presence of a Bosα-L-fucosidase resulted in a slight increase in enzymatic activity,approximately 28% at 2.5 U ml⁻¹ Coffea enzyme and 2 U ml⁻¹ Bos enzyme,over the control reactions without fucosidase.

Transglycosylation reactions have been described with eukaryoticα-D-galactosidases (Honda et al, 1990; Kitahataet al, 1992]. In anattempt to explore the possibility that reglycosylation of the Bprecursor, the H epitope, was occurring, galactose dehydrogenase and 2'fucosyllactose were added to reactions. Galactose dehydrogenase and NADwere used to oxidize and trap liberated galactose, whereas 2'fucosyllactose was added as an alternate galactosyl acceptor. It wasapparent that neither compound enhanced the rate of hydrolysis of the Bepitopes in the solid phase ELISA. Bos α-L-fucosidase was added to thereactions in an attempt to hydrolyze subterminal α1-2 fucosyl residuesand thus enhance hydrolysis of the terminal α1-3 galactose residues.

Soluble carbohydrate substrate studies

Exoglycosidase activity against five carbohydrate substrates, each in 50mM Na citrate at pH 6.0, was determined. The Coffea enzyme was moreactive against straight-chain substrates than against branchedsubstrates, with the difference in hydrolysis being roughly three-fold.The enzyme readily hydrolyzed α1-3, α1-4, and α1-6 galactosyl bonds butwith the substrates that the applicants used exhibited the highestactivity against the α1-3 linkage. Enzyme activity againstB-trisaccharide in Na citrate, MES, and PCBS was also studied. Both Nacitrate and MES increased enzymatic activity over PCBS, by 55% and 15%respectively. K_(m) studies were done using B-trisaccharide,Galα1-2Gal[Fucα1-2]β1-3GlcNAc, and melibiose in 50mM Na citrate, pH 6.0.The K_(m) values were 2,465 mM, 3,476 mM, and 1.718 mM, respectively.K_(m) in MBS and PCBS were performed using B-trisaccharide as substrate.The K_(m) in MBS and PCBS were 2.951 and 1.193, respectively.

EXAMPLE 2 DEANTIGENATION OF B EPITOPE WITH GLYCINE ENZYME

Preliminary deantigenation experiments were performed in CBS (10 mM Nacitrate+150 mM NaCl+1 mg ml⁻¹ BSA, pH 5.8), PCBS (25 mM Na citrate+60 mMNa H₂ PO₄ +75 mM NaCl+1 mg ml⁻¹ BSA, pH 5.8), and GCB (5 mM Nacitrate+300 mM glycine+1 mg ml⁻¹ BSA, pH 5.8). Incubation was for twohours at 24° C. followed by washing five times with PBS, and thentreated with neat monoclonal anti-B. As shown in FIG. 1, ten U ml⁻¹ ofenzyme incubated at hematocrit of 8% erythrocytes were most efficientlydeantigenated in GCB buffer. Complete deantigenation as measured byhemagglutination was achieved at various glycine concentrations rangingfrom 220 to 440 mM in 5 mM Na citrate buffer, pH 5.8.

Complete deantigenation as measured by hemagglutination was achieved atvarious glycine concentrations. Ten U ml⁻¹ of enzyme was incubated athematocrit of 8% B+erythrocytes in X mM Na citrate or NaH₂ PO₄ +300 mMglycine+1 mg ml⁻¹ BSA, pH 5.8 where X was tested in the range of 0 to40, for two hours at 24° C., washed five times with PBS, and treatedwith neat monoclonal anti-B. Interestingly, increasing citrate orphosphate concentrations in 300 mM glycine inhibited hydrolysis of the Bepitope (FIG. 2). Optimal citrate concentrations for deantigenation werebelow 10 mM.

Also noteworthy was the inhibitory effect of increasing concentration ofNaCl (FIG. 3). Ten U ml⁻¹ of enzyme was incubated at hematocrit of 8%B+erythrocytes in 5mM Na citrate+300mM glycine +X mM NaCl+1 mg ml⁻¹ BSA,pH 5.8 with X in the range of 0 to 40, for two hours at 24° C., washedfive times with PBS, and treated with neat monoclonal anti-B. Above 10mM NaCl significant hydrolysis of the B epitope was not detected in thehemagglutination assay.

When NaCl and glycine were combined at various concentrations to achievean osmolality of 0.310, a similar inhibitory effect was observed (FIG.4). Ten U ml⁻¹ of enzyme was incubated at hematocrit of 8% B+erythrocytes in 5 mM Na citrate +X mM glycine+Y mM NaCl+1 mg ml⁻¹ BSA,pH 5.8, where (X,Y)=(300,0), (240,30), (180,60), (120,90), (60,120),(0,150). Cells were incubated for two hours at 24° C., washed five timeswith PBS, and treated with neat monoclonal anti-B.

Deantigenation as a function of pH is shown in FIG. 5. Deantigenation inGCB could be achieved up to a pH of 6.4 at an 8% hematocrit and enzymeconcentration of 10 U ml⁻¹. Ten U ml⁻¹ of enzyme was incubated athematocrit of 8% B+ erythrocytes in 4 mM Na citrate+4 mM NaH₂ PO₄ +300mM glycine+1 mg ml⁻¹ BSA, pH=X, where X equals pHs of 5.4, 5.6, 5.8,6.0, 6.2, 6.6, 7.0, and 7.2. After two hours at 24° C., the cells werewashed five times with PBS and treated with neat monoclonal anti-B.

The effect of increasing hematocrit at a constant enzyme concentrationof 10 U ml⁻¹ is shown in FIG. 6. Ten U ml⁻¹ of enzyme was incubated athematocrit of X% B+ erythrocytes in 5 mM Na citrate +300mM glycine+1 mgml⁻¹ BSA, pH 5.8 where X%=8, 16, 24, 32 After two hours at 24° C. thecells were washed five times with PBS and treated with neat monoclonalanti-B. As shown in the graph, efficient deantigenation could beachieved at a hematocrit of 16%.

FIG. 7 illustrates the effect of increasing enzyme concentration at ahematocrit of 16%. X U ml⁻¹ of enzyme where X=1.25, 2.5, 5.0, 10.0, 20.0was incubated at hematocrit of 8% (open square) or 16% (filled diamond)B+ erythrocytes in 5 mM Na citrate+300 mM glycine+1 mg ml⁻¹ BSA, pH 5.8.After two hours at 24° C., the cells were washed five times with PBS andtreated with neat monoclonal anti-B.

FIG. 8 illustrates deantigenation as a function of time. Ten U ml⁻¹ ofenzyme was incubated at hematocrit of 8% B+ erythrocytes in 5 mM Nacitrate+300 mM glycine+1 mg ml⁻¹ BSA, pH =5.8. After t=X hours at 24° C.where X=0.25, 0.5, 1.0, and 2.0 hours, the cells were washed five timeswith PBS and treated with neat monoclonal anti-B.

Also, cells could be stored in 0.32% citrate for up to six weeks andstill be deantigenated under similar conditions. Experiments with Coffeacanephora α-D-galactosidase at low enzyme concentrations, at anyhematocrit, failed to achieve deantigenation in either PCBS, GCB, orCBS.

Albumin at a concentration of 1 mg ml⁻¹ was included in the GCB bufferbecause it was noted that the inclusion of albumin slightly reducedresidual hemolysis. Increasing concentrations of albumin to 30 mg ml⁻¹did not effect deantigenation. Identical results were obtained witheither native or heat-treated human albumin. In the transfusion art,heat-treated HSA is used since it has little immunogenicity and nopotential infectivity.

The effect of Glycine max α-D-galactosidase at a concentration of 20 Uml⁻¹ and a hematocrit of 16% on red cell indices (MCV, MCH, MCHC, RDW),ATP, 2,3 DPG, cholinesterase, osmotic fragility, hemolysis of red cells,carboxyhemoglobin, methemoglobin, % O₂ saturation, Po₂, and p50 weredetermined in PCBS and GCB. These were compared to control cellsincubated in PBS. It was evident that GCB had a limited effect onincreasing erythrocyte 2,3 DPG but had little effect on ATP and red cellcholinesterase, Table I.

Osmotic fragility of cells in GCB and PCBS were similar to thoseincubated in PBS. There was no significant change in red cell indices ineither GCB or PCBS when compared to PBS incubated cells. There was nosubstantial change in % O₂ saturation, Po₂, and p50 in either buffer;and, furthermore, there was no substantial increase in carboxy ormethemoglobin in either PCBS or GCB. In GCB, 1.53% of the red cells werehemolyzed compared to 2.16% and 1.90% in PCBS and PBS control cells,respectively.

Erythrocytes incubated in GCB maintained their native morphology asshown in FIG. 9. 16% B+cells were incubated in GCB (5 Mm Na citrate+300Mm glycine+1 mg ml⁻¹ BSA, Ph=5.8) with ten U ml⁻¹ Glycine maxα-D-galactosidase. After two hours at 24° C., the cells were washed fivetimes with PBS. The erythrocytes were then incubated with either neatmonoclonal anti-B (enzyme treated; FIG. 9A) or a 1:128 monoclonal anti-Bdilution (untreated; FIG. 9B), and photomicrographed without staining.

Additionally, extensive antigen typing was performed. The only observedchange was conversion of the B to O antigen and loss of reactivity withanti-P typing sera.

EXAMPLE 3 DEANTIGENATION OF A₂ EPITOPE

Soluble A antigens in an ELISA using type A₂ erythrocyte membranes wereused to study the activity of an α-N-acetyl-galactoaminidase from Gallusdomesticus.

Results

A microtest well coating concentration of 0.4 μg ml⁻¹ A₂ erythrocytemembranes resulted in excellent reproducibility and a sufficient signalto noise ratio. The method of Hobbs et al. (1993) was used to determineprimary and secondary antibody concentrations. Binding of anti-Adecreased with increasing enzyme concentration while UEA I bindingincreased, demonstrating the conversion of the A epitope to H epitope.Also noteworthy is the fact that, at an enzyme concentration of 5 Uml⁻¹, hydrolysis of the A epitope from A₂ membranes was virtuallycomplete. At a 1 M NaCl concentration (μ=1.01), hydrolysis of the Aepitope was decreased by 64% compared to buffer without NaCl (μ=0.01).The pH optimum of the enzyme was determined to be 3.5, with a broadactivity shoulder between pH 4 and 5. The enzyme still retained 60% ofoptimal activity at a pH of 5.8.

The effect of buffer species was also determined. 25mM MES enhanced theactivity of the enzyme, increasing hydrolysis 92.6% over PCBS.Hydrolysis of the A epitope was inversely dependent upon concentrationwith all buffer species examined. Enzyme activity in MBS was alsocompared to PCBS. The MES containing buffer increased hydrolysis ofN-acetyl-α-D-galactosamine by 42.9%.

The effect on enzyme activity of glycine, a zwitterion at pH 5.8 wasdetermined. In both NaH₂ PO₄ and MES buffers, glycine significantlyenhanced enzyme activity. No significant inhibition was evident atglycine concentrations exceeding 300 mM in a NaH₂ PO₄ buffer.

The effect of several detergents was also determined. Deoxycholic acid,cetylpyridinium chloride, and Triton X-100 (anionic, cationic, andnon-ionic detergents, respectively) inhibited enzymatic activity. CHAPS,a zwitterionic detergent, enhanced enzymatic activity.

EXAMPLE 4 FLOW CYTOMETRY ASSAY

Assay Procedure

The assay is an adaptation and different application of a previouslydescribed procedure (Sharon, 1991). Briefly, 4% suspensions of humantype B erythrocytes were incubated with exoglycosidase under a varietyof buffer conditions and enzyme concentrations as described in theresults and then washed five times with PBS (10 mM NaH₂ PO₄ +137 mMNaCl+2.1 mM KCl, pH 7.4). 100 μl of these 4% suspensions were incubatedwith 100 μl of a 1:40 dilution of monoclonal anti-B in PBS at 24° C. for30 minutes. The cells were dispersed with a 25 gauge needle and washedfive times with PBS. Next, the suspensions, 200 μl, were incubated with5 μl of neat polyclonal goat anti-mouse μ chain specific FITC conjugateat 4° C. for 30 minutes, dispersed, and washed again five times withPBS. The cell concentrations were adjusted to 1×10⁶ cells ml⁻¹, and thesuspensions dispersed before cytometry.

Cells were analyzed using a EPICS 753 flow cytometer (Coulter Cytometry,Haileah, Fla.) with a 5 W argon laser tuned at 488 nm using 150 mWoutput. Optical alignment of the instrument was obtained using 10 μm,full-bright, fluorescent polystyrene microspheres (Coulter Immunology)with coefficients of variation kept at 2% or less. Log integral greenfluorescence of 10,000 cells was collected through a 525 band-passfilter grating on forward angle light scatter versus log 90° lightscatter to exclude debris. Single parameter histograms were analyzedusing the STATS program on the MDADS II computer (Coulter Cytometrey).Data was obtained as percent fluorescent cells versus the logarithm ofrelative fluorescence. Data in the results is expressed as percentfluorescent cells as a function of the dependent variable.

Results

The 1° antibody was titrated and a 1:40 dilution was found to giveoptimal fluorescence with weaker agglutination than higher antibodyconcentrations. Fluorescence significantly decreased when less than 1 μlof the FITC conjugate was used, and 4 μl was chosen as a saturatingconcentration.

FIG. 10 shows the selective reaction of monoclonal anti-B with type Bcells and lack of reactivity with type A and O cells. 4% cell suspensionwere incubated with 1° antibody (anti-B), 2° antibody (goat anti-mouse μchain specific FITC conjugate), and the fluorescence quantitated asdescribed in the methods. Similar results were obtained with monoclonalanti-A reacting with only type A erythrocytes.

Deantigenation of type B erythrocytes as a function of enzymeconcentration is shown in FIG. 11. Four percent cell suspensions wereincubated with X U ml⁻¹ of Glycine max enzyme in 10 mM Na citrate+300 mMglycine+1 mg ml⁻¹ BSA, pH 5.8, for 30 min. at 24° C. where X=0.32, 0.63,1.25, 2.50, 5.0, & 10.00. Cells were then reacted with 1° antibody, 2°antibody FITC conjugate, and the fluorescence quantitated. At ahematocrit of 4% as little as 5.00 U ml⁻¹ of Glycine maxα-D-Galactosidase completely removed the B epitope with only a 30 minuteincubation at 24° C.

Higher enzyme concentrations were required at higher hematocrits,however, as little as 10.00 U ml⁻¹ completely removed the B epitope froma 16% suspension of type B cells. At extremely low enzymeconcentrations, 1.00 U ml⁻¹, greater than 94% of detectable fluorescencewas removed from a 4% cell suspension after a two hour incubation at 24°C. (FIG. 12). Four percent cell suspensions were incubated with 0.20 Uml⁻¹ of Glycine max enzyme in 10 mM Na citrate+300 mM glycine+1 mg ml⁻¹BSA, pH 5.8, for X minutes at 24° C. where X=0, 15, 30, 60, and 120minutes. They were then reacted with 1° antibody, 2° antibody FITCconjugate, and the fluorescence quantitated.

Deantigenation as a function of pH is shown in FIG. 13. Four percentcell suspensions were incubated with 1.25 U ml⁻¹ of Glycine max enzymein 5 mM Na citrate+5 mM Na H₂ PO₄ +300 mM glycine, pH=X, for 30 minutesat 24° C. where pH X =5.4, 5.8, 6.2, 6.6, 7.0; & 7.4. They were thenreacted with 1° antibody, 2° antibody FITC conjugate, and thefluorescence quantitated. At an enzyme concentration of 1.25 U ml⁻¹,more than 99% of the detectable B antigen was removed at pH 5.4 with a30 minute incubation at 24° C. Complete deantigenation could be achievedat higher pHs with higher enzyme concentrations; for example, a 16% cellsuspension could be deantigenated at pH 6.2 with 10.00 U ml⁻¹ of enzyme.

The effect of buffer composition was studied. FIG. 14 shows removal ofthe B epitope in three different buffers: PCBS (60 mM NaH₂ PO₄ +25 mM Nacitrate+75 mM NaCl+1 mg ml⁻¹ BSA, pH 5.8), GCB (5 mM Na citrate+300 mMglycine+1 mg ml⁻¹ BSA, pH 5.8), or CBS (10 mM Na citrate+140 mM NaCl,+1mg ml⁻¹ BSA, pH 5.8). Four percent cell suspensions were incubated with5.0 U ml⁻¹ of Glycine max enzyme in each of the three buffers for twohours at 24° C. The cells were developed with 1° antibody, 2° antibodyFITC conjugate, and the fluorescence quantitated. It was evident that atlow enzyme concentrations and low hematocrits efficient deantigenationwas only achieved in GCB. These findings correlated well with cellsuspension studies using conventional hemagglutination assays undersimilar assay conditions.

The effect of various isosmolal solutions of NaCl and glycine werestudied, FIG. 15. Four percent cell suspensions were incubated with 1.5U ml⁻¹ of Glycine max enzyme in 10 mM Na citrate+X mM glycine, +Y mMNaCl+1 mg ml⁻¹ BSA, pH 5.8, for 30 minutes at 24° C. where(X,Y)=(0,129), (52,103), (103,77), (173,52), (206,26), (258,0). Thecells were reacted with 1° antibody, 2° antibody FITC conjugate, andthen fluorescence quantitated. It was evident that increasingconcentrations of NaCl inhibited deantigenation. Similar findings wereconfirmed in conventional hemagglutination assays.

EXAMPLE 5 CLONING AND SEQUENCING OF SOYBEAN α-D-GALACTOSIDASE

Cloning of Soybean α-D-Galactosidase:

A soybean cDNA library (a gift from Joe Polacco, University ofMissouri), made in lambda ZAP (Stratagene), was screened under lowstringency hybridization and wash conditions with a radiolabeled portionof the pinto bean α-D-galactosidase gene (SEQ ID No.:1) The positiveclone obtained, SB-10, was excised from the lambda vector and sequenced.The deduced amino acid sequence was compared to that of the guar(Overbeek et al., 1989) and pinto bean α-D-galactosidases. SB-10 was notfull length and was missing the expected 5' end of the gene,corresponding to the start codon, signal peptide, and mature N-terminus.

Modern soybean, Glycine max, is an allotetraploid that formed from theunion of two genetically distinct species. This means that in additionto the genetic diversity bred into modern soybean for agronomic reasons,it is also likely that there are two or more different copies of theα-D-galactosidase gene in soybean in any given soybean cultivare. Aswith corn, soybean is a crop planted in many climates and soilconditions, Many strains exist with different characteristics for growthunder these conditions. This genetic diversity may be reflected in theamino acid sequence for soybean α-D-galactosidase.

The SB-10 clone Eco RI insert was radiolabeled and used as a probe tore-screen the lambda ZAP::SB cDNA library under high stringencyhybridization and wash conditions. A full length clone, SB-14a, waspurified and excised. The SB- 14a clone was sequenced (SEQ ID No.:2) andfound to be 1750 nt in length, with a 1266 nt open reading frameencoding a protein with a 55 amino acid signal peptide (SEQ ID No.:3)and a mature length of 363 amino acids (SEQ ID No. 4). Identity of theSB-14a cDNA clone as the gene encoding soybean α-D-galactosidase wasconfirmed by comparison of the deduced amino acid sequence of the cloneto the N-terminal and cyanogen bromide-derived peptide sequencesobtained from the native soybean α-D-galactosidase. Comparison of thededuced amino acid sequence of the SB-14a clone to that of otherreported α-D-galactosidases and α-N-acetylgalactosaminidases showed ahigh degree of sequence similarity between the different proteins.

Expression of the Soybean α-D-Galactosidase Gene in Pichia:

Active recombinant soybean α-D-galactosidase was obtained with thePichia Expression Kit (Invitrogen) using the Pichia expression/secretionvectors pHILS1 and pPIC9. Polymerase chain reaction was used to amplifythe coding region of SB-14a. The 5' PCR primer corresponded to themature N-terminus of the protein and contained an Eco RI site designedto allow cloning of the PCR product such that the soybean ORF wasin-frame with the translation start codon provided by the Pichiaexpression vectors. An oligo annealing to M13-pUC was used as the 3' PCRprimer. The PCR product was digested with Eco RI and cloned into the EcoRI site of pHILS1 and pPCI9. The vector-insert junctions of theseconstructs were sequenced to determine proper ligation, orientation, andmaintenance of reading frame.

Two clones, pHILS1/SB229 and pIC9/SB217, were chosen for expression inthe Pichia system. Midi-prepped plasmid DNA from each clone walinearized by Aat II/Tth 111I digestion. Pichia spheroplasts weretransformed with these linear constructs. Greater than fifty His+transformants were obtained for each construct. These were screened forthe His+ Mut⁻ phenotype, which indicates correct integration of thesoybean construct into the yeast chromosome. Those transformants foundto be correctly integrated were grown and induced on a small scale andthe culture media was assayed for α-D-galactosidase activity. Thetransformant found to have the highest activity, pHILS1/SB229-32, wasused for a medium scale induction and expression of the recombinantsoybean α-D-galactosidase enzyme.

Throughout this application various publications are referenced bycitation or number. Full citations for the publications referenced bynumber are listed below. The disclosures of these publications in theirentireties are hereby incorporated by reference into this application inorder to more fully describe the state of the art to which thisinvention pertains.

The invention has been described in an illustrative manner, and it is tobe understood that the terminology which has been used is intended to bein the nature of words of description rather than of limitation.

Obviously, many modifications and variations of the present inventionare possible in light of the above teachings. It is, therefore, to beunderstood that within the scope of the appended claims, the inventionmay be practiced otherwise than as specifically described.

                  TABLE I                                                         ______________________________________                                        Effect of buffer composition on erythrocyte ATP,                              2,3 DPG, and cholinesterase                                                                               PBS                                                        GCB        PCBS    control                                           ______________________________________                                        ATP         98%         116%    100%                                          2,3 DPG    119%         81%     100%                                          cholinesterase                                                                           102%         87%     100%                                          ______________________________________                                         B+ erythrocytes were incubated in the designated buffer. After 2 hr at        24° C., the cells were washed five times with PBS and assayed for      the indicated analyte. All data points are the mean of four independent       determinations and expressed as a % of the PBS buffer control.           

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We claim:
 1. A method of preparing deantigenated erythrocytes includingthe steps ofisolating erythrocytes of a defined blood type; suspendingthe isolated erythrocytes in zwitterionic buffer wherein thezwitterionic buffer consists essentially of 0.1 to 20 mM Na titrate, 220to 440 mM glycine or alanine and 0.01 to 30 mg ml⁻¹ albumin at pH 5.8;adding a multimeric eucaryotic exoglycosidase with multiple subunitsselected to act on the isolated erythrocytes; incubating at 24° C. forbetween one and two hours; and washing in phosphate buffered saline. 2.A method according to claim 1 wherein the erythrocytes are selected fromthe group consisting of blood types A₂, B and A₂ B.
 3. A methodaccording to claim 1 wherein the zwitterionic buffer consists of 5 mM Nacitrate, 300 mM glycine and 1 mg ml⁻¹ albumin at pH 5.8.
 4. A methodaccording to claim 1 wherein the erythrocytes express B antigen and theexoglycosidase is Glycine max α-D-galactosidase.
 5. A method accordingto claim 1 wherein the erythrocytes express A₂ antigen and theexoglycosidase is Gallus domesticus α-N-acetyl-galactosaminidase.
 6. Adeantigenation buffer consisting of 0.1 to 20 mM Na citrate, 220 to 440mM glycine and 0.1 to 30 mg ml⁻¹ albumin at pH 5.8.
 7. A deantigenationbuffer according to claim 6 consisting of 5 mM Na citrate, 300 mMglycine and 1 mg ml⁻¹ albumin at pH 5.8.